Critical periods of the mitotic cycle: influence of aminopterin and thymidine on production of chromosomal aberrations by radiation in Crepis capillaris.

The influence of aminopterin (AP), tritiated thymidine ([3H] TdR) and "cold" thymidine (TdR) on production of chromosomal aberrations in meristematic cells of Crepis capillaris irradiated in different stages of the mitotic cycle with 300 rad of 63Co gamma-rays was studied. All the chemical treatments increased most of all the frequency of aberrations induced during two "critical periods" localized before the stage of DNA synthesis (fixation 9 h after irradiation) and before that of mitosis (4 h). Treatments with TdR and [3H]TdR increased most of all the frequency of chromatid aberrations when irradiation was performed in G1, and the frequency of gaps when irradiated in G2. Treatment with AP increased the yield of different types of aberration more uniformly. The modifying effect of the chemicals tested appeared to be independent of replicative synthesis. The "critical periods" are suggested to be the stages when regular "proof reading" and correction of spontaneous errors takes place [9,13]. In addition to this regular mechanism, radiation induces an "emergency" mechanism of repair. AP inhibits the mechanism of regular repair; in addition TdR and [3H] TdR suppress the lateral spread of primary injuries across the chromosome.     

Sodium fluoride-induced chromosome aberrations in different stages of the cell cycle: a proposed mechanism

In an attempt to clarify the controversy about sodium fluoride (NaF) clastogenicity, the induction of chromosome aberrations in Chinese hamster ovary cells (CHO) by NaF was investigated. Following a protocol used for screening chemicals for clastogenic activity, significant increases of aberrant cells were observed when cells were exposed to NaF for 4 h and harvested 8 h later. Cell-cycle kinetic studies demonstrated most cells were exposed in G2 of the cell cycle. Smaller increases in aberrant cells were observed when cells were harvested 20 h later (most cells were exposed in G1/S). The sensitivity of G2 cells to NaF was investigated further, along with the induction of aberrations at low doses. The results indicated that G2 cells are sensitive to NaF and the percent of aberrant cells increased with dose and length of exposure. With a 3-h exposure until harvest, no statistically significant increase in aberrant cells was observed at doses below 10 micrograms/ml NaF. These data are consistent with a threshold for NaF-induced clastogenicity around 10 micrograms/ml, as has been proposed previously (Scott and Roberts, 1987). It thus may be predicted that clastogenic effects would not occur in humans exposed to the levels of fluoride that are present in drinking water or dentifrices. An understanding of the mechanism of NaF-induced clastogenicity would help to clarify this point. It has previously been reported that NaF inhibits DNA synthesis/repair. The types of aberrations, mostly deletions and gaps, the induction of endoreduplicated cells, the cell-cycle delay and the sensitivity of G2 cells to NaF observed are similar to that reported in the literature for DNA synthesis/repair inhibitors like aphidicolin (APC). Similarities in the induction of aberrations by NaF and APC were confirmed in experiments with G2 cells. Based on these results and those previously reported for NaF and APC, it is proposed that NaF-induced aberrations may occur by an indirect mechanism involving the inhibition of DNA synthesis/repair.     

Formation of chromosome aberrations in human lymphocytes to the action of 5-fluorodeoxyuridine used separately and in combination with gamma irradiation: changes in the effect in the course of the mitotic cycle

Human lymphocytes were treated after different times of incubation, either by 60Co gamma-rays (1 Gy) followed by 5-fluorodeoxyuridine (FUdR, 2.10-7 M during 2,5 h) or by radiation and FUdR, separately. Chromosomal aberrations were studied after 51 h of incubation. When administered alone, FUdR increased the frequency of chromatid aberrations and gaps over the spontaneous level. This increase took place mainly during two periods of the mitotic cycle, namely, on the borderline between G1 and S stages and at the end of the G2 stage. FudR barely affected the frequency of chromosomal aberrations. THe effect did not depend upon the concentration of FUdR. Irradiation during the G1 stage produced chromatid aberrations and gaps with the same frequency as FUdR, whereas the frequency of chromosome aberrations was much higher. When administered after irradiation, FUdR increased the frequency of all types of aberrations; the periods of mitotic cycle when this increase was statistically significant correspond to those of "mutagenic" action of FUdR mentioned above. This pattern may be easily explained if one postulates that in our experiments FUdR exhibited the features of a "pseudomutagen" i.e. the factor which suppresses repair of primary lesions (spontaneous or radiation-induced) without giving rise to new mutational changes.     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Possible mechanisms of the occurrence of chromosome aberrations. I. Patterns in the occurrence of aberrations induced by ultraviolet irradiation

The frequency of chromosome aberrations induced by UV light at wavelengths 254, 265, 280 and 302 using doses 2-10 J/m2 in the primary culture of mouse embryonic fibroblasts during the G1, S and G2 phases was studied at metaphase of the first mitosis. Two classes of chromosome aberrations were distinguished. These classes differ in the time intervals of the final establishment of the cell cycle. The aberrations of the class 1 emerge before the beginning of prometaphase (possibly, at interphase). Formation of the second class aberrations is completed during the metaphase. It is shown that the class 1 aberrations occur with almost the same rate in approx. 7% of cells, irrespective of the cell cycle, irradiation dose and wavelength. It is suggested that these aberrations arise as a result of indirect UV action on the chromosome structures; the mechanism of their emergence does not depend on DNA replication. The class 2 aberrations do not appear after UV irradiation during the post-DNA-synthetic G2 phase of the cell cycle. However, after UV treatment at the G1 or S periods, they represent the majority of aberrations and their rate increases almost monotonously with the radiation dose. The UV action spectrum for these aberrations coincides with the adsorption spectrum of thymidine and the action spectrum for DNA cross-links. Thus, it may be inferred that formation of DNA cross-links following thymine dimerization is the first step in formation of UV-induced aberrations of the class 2. The passage of cells through DNA replication is a very important step in the process of their emergence.     

Study on the repair of the radioinduced lesions involved in the formation of chromosomal aberrations in G0 human lymphocytes after exposure to gamma-rays and fast neutrons

Cytosine arabinoside (ara-C), an inhibitor of DNA synthesis and repair, has been used to study the mechanisms of formation of chromosomal aberrations after exposure to low- and high-LET radiation. When G0 human lymphocytes were exposed either to gamma-rays or to d(50 MeV)-Be neutrons and immediately treated with ara-C for increasing periods of time, the frequency of aberrations (dicentrics) increased sharply. For gamma-rays, the enhancement increased with the duration of the treatment up to 5 h, whereas for neutrons, an ara-C treatment lasting for 5 h was no more effective than treatment for 3 h. These results were confirmed by the second experiment in which ara-C was administered for 3 h with an increasing time delay following irradiation. Since no increase in the dicentric frequency was observed when ara-C was administered 5 h after gamma-irradiation, it is suggested that the induced breaks rejoined within that time. For neutrons, the data were conflicting since the repair was completed within 3 h after a dose of 0.5 Gy, and in approximately 5 h after a dose of 2.0 Gy. From both experiments, it appears that gamma-rays and fast neutrons produce similar types of lesions, as ara-C increased the frequencies of aberrations induced by both types of radiation. However, the ara-C treatment resulted in a smaller increase in aberrations following neutron irradiation. According to the enzymatic nature of break formation and the mode of action of ara-C on the polymerase activity, it is suggested that, in addition to double-strand breaks, single-strand breaks could be the lesions involved in the repair processes inhibited by ara-C. Single-strand breaks formed directly or by secondary reactions would, therefore, be one of the major lesions responsible for the aberrations produced by gamma and neutron radiations.     

Relationship of DNA repair to chromosome aberrations, sister-chromatid exchanges and survival during liquid-holding recovery in X-irradiated mammalian cells

The repair of X-ray induced DNA single strand breaks and DNA—protein cross-links was investigated in stationary phase, contact-inhibited mouse cells by the alkaline-elution technique. Approx. 90% of X-ray induced single strand breaks were rejoined during the first hour of repair, whereas most of the remaining breaks were rejoined more slowly during the next 5 h. At early repair times, the number of residual non-rejoined sungle strand breaks was approx. proportional to the X-ray dose. DNA—protein cross-links were removed at a slower rate (T1/2 approx. 10–12 h). Cells were held in stationary growth for various periods of time after irradiation before subculture at low density to score for colony survival (potentially lethal damage repair), chromosome aberrations in the first mitosis, and sister-chromatid exchanges in the second mitosis. Both cell killing and the frequency of chromosome aberrations decreased during the first several hours of recovery, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA-strand breaks. Relatively few sister-chromatid exchanges were observed when the cells were subcultured immediately after X-ray. The exchange frequency rose to maximum levels after a 4-h recovery interval, and returned to control levels after 12 h of recovery. The possible relationship of DNA repair to these changes in survival, chromosome aberrations, and sister-chromatid exchanges during liquid-holding recovery is discussed.     

The synergistic effect of aphidicolin on the yield of X-ray-induced chromosome aberrations throughout the cell cycle in JU56 cells

The effect of a 2-h post-treatment with aphidicolin at a dose sufficient to inhibit DNA synthesis on the yield of X-ray-induced chromosomal aberrations throughout the cell cycle was measured. Exposure to aphidicolin during and after irradiation brought about an increase in exchanges in cells irradiated in G2, in sister unions only in cells irradiated in S, and in all chromosome aberration types (fragments, sister unions, and dicentrics) in cells irradiated in G1. It is suggested that, during G1 and G2 but not during S inhibiting the repair enzyme alpha-polymerase brings about the conversion of some X-ray-induced DNA lesions to double-strand which can then take part in aberrations.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VII. Complementation analysis of X-irradiated wild-type CHO-K1 and xrs mutant cells using the premature chromosome condensation technique

The complementation effect of wild-type CHO-K1 and xrs mutants after fusion, as judged by the frequencies of X-ray-induced G1 and G2 premature chromosome condensation (PCC), was studied. For induction of PCC, X-irradiated interphase cells (G1 and G2) were fused immediately with untreated mitotic cells of the same cell line or with mitotic cells of another line. The frequencies of breaks in G1-PCC, or breaks and chromatid exchanges in G2-PCC were determined and the latter parameter was compared with the frequency of chromosomal aberrations in mitotic cells following G2 irradiation. CHO-K1 cells were capable of complementing the X-ray sensitivity of both xrs 5 and xrs 6 cells. However, full restoration of the repair defect in xrs cells could never be accomplished. The mutants failed to complement each other. In CHO-K1 cells, the incidence of chromosomal aberrations was significantly higher in G2-PCC (2.5-fold) than that observed in mitotic cells at 2.5 h after irradiation. The ratio of the induced frequency of aberrations in G2-PCC to that in mitotic cells was correlated with the degree of repair of DNA double-strand breaks (dsb) and reached almost 1 in xrs 5 cells indicating no repair. In addition the data indicated that, during the period of recovery of CHO-K1 cells, X-ray-induced breaks decreased but exchanges remained at the same level. In contrast, due to a deficiency in rejoining of dsb in xrs mutants, breaks remained open for a long period of time, allowing the formation of additional chromatid exchanges during recovery time.     

DNA-repair kinetics and the sensitivity of cells to X-ray-induced chromosome aberrations: a mouse myeloid leukemia cell line and normal mouse bone marrow cells

The frequency of X-ray-induced chromosome aberrations in G1 ML-1 mouse myeloid leukemia cells and normal mouse bone marrow cells increased with post-irradiation incubation with the DNA-repair resynthesis inhibitor 1-beta-D-arabinofuranosylcytosine (araC), but the frequency of aberrations in the leukemic cells increased with quite a different time response compared to the normal cells. Irradiated normal mouse bone marrow cells had a rapid increase in the frequency of chromosome exchanges and deletions with increasing araC incubation time, for example, an increase was observed with 0.5 h araC incubation. In contrast, the ML-1 cells did not have a significant increase in aberrations until 1-2 h post-irradiation incubation with araC. These results suggest that the ML-1 cells, per unit time, initially undergo less repair of the X-ray-induced DNA damage that can be converted into chromosome aberrations. We previously showed that the ML-1 cells have a higher frequency of X-ray-induced chromosome aberrations compared to normal cells and the results presented here indicate that a slower rate of repair resynthesis is contributing to the increased sensitivity of the ML-1 cells.     

A comparison of the roles of p53 mutation and AraC inhibition in the enhancement of bleomycin-induced chromatid aberrations in mouse and human cells

Previous studies have shown that p53 is involved in the repair of bleomycin-induced DNA damage, and that the frequency of bleomycin-induced chromatid aberrations is elevated in G(2)-treated p53 null transgenic mouse embryo fibroblasts (MEF) as compared to isogenic controls. To further characterize p53-mediated DNA repair, we studied the effect of p53 status on the ability of the DNA repair inhibitor 1-ss-D-arabinofuranosylcytosine (AraC) to sensitize MEF to bleomycin-induced chromatid aberrations. Both p53+/+ and p53-/- MEF were treated in G(2) with 0 to 7.5 microg/ml bleomycin in the presence or absence of AraC (5x10(-5) M). The frequency of bleomycin-induced chromatid aberrations was significantly higher in p53-/- cells than wild-type cells in the absence of AraC. AraC treatment significantly increased the frequency of bleomycin-induced chromatid aberrations in p53+/+ MEF to the levels in p53-/- (no AraC) but had no effect in p53-/- MEF. These results suggest that an AraC-sensitive DNA repair component is altered or absent in p53-/- cells. Similar results were observed in p53-mutant WTK1 and wild-type TK6 human lymphoblast cells exposed to 0 to 3 microg/ml bleomycin in G(2). However, AraC did cause a small increase in bleomycin sensitivity in WTK1 cells. This difference from the p53-/- MEF response may be due to differences in p53-mutant phenotype. To determine whether mutation of p53 alters DNA replication fidelity, p53+/+ and p53-/- MEF were exposed to 0 to 1 microg/ml mitomycin C (MMC). MMC did not induce chromosome aberrations in either cell line treated in G(2) but did with the same effectiveness in both cell lines treated in S-phase. Thus, p53 deficiency does not affect DNA replication fidelity or the repair of MMC-induced DNA damage.     

Effects of repair inhibition in the G1 phase of clastogen-treated human lymphocytes on the frequencies of chromosome-type and chromatid-type aberrations and sister-chromatid exchanges

It has been shown that certain types of DNA lesions induced by an S-dependent clastogen are converted to chromosome-type aberrations when their repair is inhibited in the G1 phase of the cell cycle. The purpose of the present study was to investigate which kinds of repair inhibitors have the ability to induce chromosome-type aberrations in cells having DNA lesions and which kinds of DNA lesions will be converted to chromosome-type aberrations when their repair is inhibited. For this purpose, human peripheral blood lymphocytes, which were treated with a clastogen in their G0 phase, were post-treated with one of several kinds of repair inhibitors in the G1 phase, and resulting frequencies of both chromosome-type and chromatid-type aberrations as well as of sister-chromatid exchanges (SCEs) were compared with those of the control cultures: chromatid-type aberrations and SCEs were adopted as cytogenetic indicators of lesions remaining in S and G2 phases. Chemicals used for the induction of DNA lesions were 4-nitroquinoline 1-oxide (4NQO), methyl methanesulfonate (MMS) and mitomycin C (MMC); inhibitors used were excess thymidine (dThd), caffeine, hydroxyurea (HU), 5-fluoro-2'-deoxyuridine (FdUrd), 1-beta-D-arabinofuranosylcytosine (ara C), 9-beta-D-arabinofuranosyladenine (ara A), 1-beta-D-arabinofuranosylthymine (ara T) and aphidicolin (APC). Induction of chromosome-type aberrations was observed in cells pretreated with 4NQO or MMS followed by ara C, ara A, ara T or APC, whereas other combinations of a clastogen and an inhibitor did not induce them. Among the inhibitors, ara C alone induced chromosome-type aberrations in cells without pretreatment. Chromatid-type aberrations were increased only in cells pretreated with MMC and their frequency was enhanced further by post-treatment with ara C. All of the clastogens used in the present experiments induced SCEs. Most inhibitors did not modify the SCE frequencies except for ara C which synergistically increased the frequency in MMC-treated cells. The present study offers further evidence that the lesions responsible for chromosome-type aberrations are those which are repaired quickly, and that they are converted to chromosome-type aberrations when repair by polymerase alpha is inhibited. The effects of ara C on MMC-induced lesions are considered residual effects of ara C treatment in the S or G2 phases rather than repair inhibition in the G1 phase.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. IV. Study of chromosomal aberrations and sister-chromatid exchanges by restriction endonucleases and inhibitors of DNA topoisomerase II

Induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) was studied in wild-type Chinese hamster ovary (CHO-K1) cells and its 2 X-ray-sensitive mutants xrs 5 and xrs 6 (known to be deficient in repair of DNA double-strand breaks (DSBs] by restriction endonucleases (REs) and inhibitors of DNA topoisomerase II known to induce DNA strand breaks. Five different types of REs, namely CfoI, EcoRI, HpaII (which induce cohesive DSBs), HaeIII and AluI (which induce blunt DSBs) were employed. REs that induce blunt-end DNA DSBs were found to be more efficient in inducing chromosomal aberrations than those inducing cohesive breaks. xrs 5 and xrs 6 mutants responded with higher sensitivity (50-100% increase in the frequency of aberrations per aberrant cell) to these REs than wild-type CHO-K1 cells. All these REs were also tested for their ability to induce SCEs. The frequency of SCEs increased in wild-type as well as mutant CHO cells, the induced frequency being about 2-fold higher in xrs mutants than in the wild-type cells. We also studied the effect of inhibitors of DNA topoisomerase II, namely 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposid (VP 16), at different stages of the cell cycle of these 3 types of cells. Both drugs increased the frequency of chromosomal aberrations in G2 cells. The mutants showed increased sensitivity to m-AMSA and VP 16, xrs 6 cells being 10- and 2-fold more sensitive than wild-type CHO-K1 cells respectively, and xrs 5 responding with 2-fold higher sensitivity than xrs 6 cells. G1 treatment of CHO cells with m-AMSA increased both chromosome- and chromatid-type aberrations, xrs mutants being about 3-fold more sensitive than CHO-K1 cells. The frequency of SCEs increased also after treatment of exponentially growing and S-phase CHO cells with m-AMSA and the higher sensitivity of xrs mutants (2-fold) was evident. The S-phase appeared to be a specific stage which is most prone for the induction of SCEs by m-AMSA. The results indicate that DNA DSBs induced by REs and inhibitors of DNA topoisomerase II correlate closely with induced chromosomal aberrations and SCEs in these cell lines, indicating that DSBs are responsible for the production of these 2 genetic endpoints.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Formation of chromatid-type aberrations in G2 stage of the cell cycle

CHO cells were treated in G₁ stage of the cell cycle with chromosome-breaking agents that act in an S-dependent manner. The cells were challenged in G₂ stage, before fixation, with various inhibitors of DNA synthesis or repair. Short-wave UV, mitomycin C, decarbomyl mitomycin and 4-nitroquinoline oxide (4NQO) were used as chromosome-breaking agents. The inhibitors of DNA repair or synthesis used were hydroxyurea, aphidicolin and caffeine. Permeabilization of cells followed by a treatment with Neurospora endonuclease (a treatment to convert DNA single-strand breaks into double-strand breaks) did not have any influence on the frequencies of chromatid aberrations induced by the chemicals used, whereas with the inhibitors the extent of potentiation varied depending on the mutagen and the inhibitor used.     

Suppressive effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in the G1 phase of human lymphocytes

1-beta-D-Arabinofuranosylcytosine (ara C) induces chromosome-type aberrations in mammalian cells by inhibiting repair replication in the G1 phase. The effect of novobiocin, an inhibitor of prokaryotic gyrases, on G1 repair in human cells was studied cytogenetically using this characteristic of ara C. The experiment was based on the assumption that if novobiocin inhibits the relaxation of chromatin required prior to repair replication, it would reduce the frequency of chromosome-type aberrations in cells treated with a mutagen followed by posttreatment with ara C. It has also been shown that in lymphocytes ara C induces chromosome-type aberrations which were not caused by any induced DNA lesion, and that the frequency of these aberrations changes with the age of the blood donor. The effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in lymphocytes without mutagen pretreatment was also investigated for blood samples from donors of different ages. Human peripheral blood lymphocytes, which were either untreated of treated with 4-nitroquinoline-N-oxide (4NQO) or methyl methanesulfonate (MMS), were posttreated in their early G1 phase with ara C only or ara C and novobiocin. The resulting chromosome-type aberrations were observed in cells in their first mitoses, and a comparison was made between the frequency of aberrations occurring in the presence of novobiocin and in its absence. The results showed that novobiocin reduced the frequency of chromosome-type aberrations induced by ara C in both mutagen-pretreated and -non-pretreated cells, and that lymphocytes from younger donors were less sensitive to novobiocin. The present study demonstrated cytogenetically the existence of a novobiocin-sensitive process to induce chromosome recombination in G1 lymphocytes.     

A quantitative comparison of potentially lethal damage repair and the rejoining of interphase chromosome breaks in low passage normal human fibroblasts

After long postirradiation incubation periods, the residual frequency of prematurely condensed chromosome fragments following X-ray exposure of noncycling diploid human fibroblasts was found to be correlated with the frequency of chromosome aberrations observed under identical treatment conditions when the cells were subcultured and scored after they reached mitosis. Over a wide range of doses, the proportion of such cells without aberrations at their first metaphase was not significantly different from the proportion able to form macroscopic colonies. Further, the rate of rejoining of interphase chromosome breaks was the same as the rate of increase in survival due to the repair of potentially lethal damage (PLD). These results suggest that there is a one-to-one correspondence between the initial breakage and rejoining of G0 chromosomes and the induction and repair of PLD measured by delayed plating from plateau-phase cultures of these cells.     

Potentiating effects of organomercuries on clastogen-induced chromosome aberrations in cultured Chinese hamster cells

Mercury compounds are among the most serious environmental pollutants. In this communication, the potentiating effects of organic and inorganic mercuries on clastogen-induced chromosome aberrations were studied in Chinese hamster CHO K1 cells. Post-treatment with monoalkylated mercuries — methyl mercuric chloride (MeHgCl) and ethyl mercuric chloride (EtHgCl) - increased the number of breakage-and exchange-type aberrations induced by 4-nitroquinoline 1-oxide (4NQO) and methyl methanesulfonate. With the DNA crosslinking agents mitomycin C (MMC) and cisplatin, MeHgCl enhanced both types of aberrations while EtHgCl enhanced breakage-type aberrations only. Since these monoalkylated mercuries did not show clastogenic effects by themselves under the present experimental conditions, the increases in chromosome aberrations were not additive. Dialkylated mercuries (dimethyl mercury and diethyl mercury) and inorganic mercuries (HgCl and HgCl₂) did not show any potentiating effects.

When MMC- or 4NQO-treated cells were post-treated with MeHgCl during the G1 phase, both breakage- and exchange-type aberrations were enhanced. Treatment with EtHgCl during the G1 phase also enhanced both types of aberrations induced by 4NQO. With MMC, however, G1 treatment with EtHgCl did not show any potentiating effect. MeHgCl and EtHgCl treatments during the G2 phase enhanced breakage-type aberrations only.

Based on these results, the following possible mechanisms for potentiation of clastogenicity by monoalkylated mercuries were suggested; (1) they interfere with repair of base lesions induced by 4NQO and MMS during the pre-replicational stage, thereby increasing unrepaired DNA lesions which convert into DNA double-strand breaks in S phase, (2) MeHgCl (but not EtHgCl) also inhibits repair of crosslinking lesions during the pre-replicational stage, and (3) their G2 effects enhance breakage-type aberrations only.     

Quantitative analyses of the induction of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed to gamma-rays and mitomycin-C in combination

Most chemicals are S-dependent and are potent inducers of SCE, but do not produce chromosome-type aberrations in the first metaphases after exposure. Ionizing radiation, which is an S-independent agent, produces chromosome-type aberrations, especially dicentrics and rings, but inefficiently produces chromatid-type aberrations. A series of experiments has been performed to investigate whether cytogenetic damage induced by ionizing radiation (gamma-rays) might be assessed separately from that induced by the alkylating chemical, mitomycin C (MMC), when human lymphocytes were exposed to these 2 agents in combination. Whole-blood cultures of human lymphocytes in G0 phase were exposed to gamma-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA), analyzed in cultures incubated for 56 h without BrdUrd, and sister-chromatid exchanges (SCEs) in cultures incubated for 72 h with BrdUrd. The frequency of chromosome-type aberrations (dicentrics and rings) increased with increasing doses of gamma-rays from 0.5 to 4.0 Gy. The dose-response relationships were the same with or without concomitant treatment with MMC (10(-6) M). Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and gamma-rays (2 Gy). There was no interaction between MMC and gamma-rays concerning these 2 endpoints.     

Chromatid damage induced by fluorescent light during G2 phase in normal and Gardner syndrome fibroblasts. Interpretation in terms of deficient DNA repair

Skin fibroblasts from Gardner syndrome (GS) compared with those from normal donors showed a significantly higher incidence of chromatid gaps and breaks following exposure to low-intensity, cool-white fluorescent light during G2 phase of the cell cycle. Considerable evidence supports the concept that chromatid gaps and breaks seen directly after exposure to DNA-damaging agents represent unrepaired DNA single- and double-strand breaks respectively. The changes in incidence of chromatid aberrations with time after light exposure are consistent with the sequence of events known to follow DNA damage and repair. Initially, the incidence of light-induced chromatid gaps was equivalent in GS and normal fibroblasts. In the normal cells, the chromatid gaps disappeared by 1 h post-exposure, presumably as a result of efficient repair of DNA single-strand breaks. In contrast, the incidence of gaps increased in GS cells by 0.5 h followed by a decrease at 1 h and concomitant increase in chromatid breaks. It appears from these findings that the increased incidence of chromatid damage in GS fibroblasts results from deficient repair of DNA single-strand breaks which arise from incomplete nucleotide excision of DNA damage during G2 phase.